Efficient transport of the mRNA is an important step for achieving efficient expression. RNA transport is controlled by a complex interaction of cellular proteins with the mRNAs in the nucleus, leading to cytoplasmic export and expression. Two types of potent retroviral RNA transport elements have been identified. The constitutive transport element (CTE) is essential for the nucleo-cytoplasmic export of unspliced RNA of the simian type D retroviruses. (Bray, et al., Proc Natl Acad Sci USA 91:1256-1260, 1994; Tabernero, et al., J Virol 71:95-101, 1997; Tabernero, et al., J Virol 70:5998-6011, 1996). A related element was identified in a subclass of rodent intracisternal A particle retroelements (Tabernero, et al., J Virol 71:95-101, 1997). The RNA transport element (RTE) is present in another subset of rodent intracisternal A particle retroelements (Nappi, et al., J Virol 75:4558-69 2001). The primary sequence of these elements are distinct. It has been demonstrated that both CTE and RTE are posttranscriptional control elements promoting expression of otherwise poorly expressed mRNAs such as gag and env from a mutant HIV lacking the viral Rev-RRE control mechanism (Nappi, et al., J Virol 75:4558-69 2001; Valentin, et al., J Virol 71:9817-9822, 1997; von Gegerfelt & Felber. Virology 232:291-299, 1997)
CTEs and RTEs are RNA binding sites for distinct cellular factors mediating the export of CTE- and RTE-containing mRNAs. RTEs and CTEs fold into distinct extended RNA stem-loop structures. Both CTEs and RTEs function in cells of different species, indicating conserved cellular export mechanisms. While the cellular TAP/NXF1 has been identified to be the CTE export factor (Grüter, et al., Mol Cell 1:649-659, 1998) the cellular factor promoting RTE export is still unknown. TAP/NXF1 does not bind RTE (Nappi, et al., J Virol 75:4558-692001). However, using a microinjected Xenopus oocyte RNA transport assay, it was found that CTE RNA can compete for RTE export, indicating, that TAP/NXF1 is involved in RTE RNA export. Not to be bound by theory, the current working model implies another factor interacting with RTE and acting as adapter to link RTE to the export receptor TAP/NXF1.
As noted above, an important step in achieving efficient expression in eukaryots is RNA transport. The role of CTEs and RTEs in RNA transport has been studied individually. The presence of more than one copy of CTE in some, but not all, cases has shown increases in expression relative to one copy of CTE alone (Wodrich et al Nucl Acid Res 28:901-910, 2000). However, the effects of the combination of CTE and RTE have not been evaluated.
This invention provides a novel tool to improve expression of unstable mRNAs. The invention provides a modified RTE element that functions better than the wildtype element. Moreover, the invention provides vectors comprising a combination of an RTE, e.g., the mutant RTE, and a CTE, which combination enhances gene expression. Since the function of these elements is conserved in mammalian cells, the use of the invention (RTE-CTE combination of RNA export elements) provides a simple method to improve gene expression to levels otherwise only achieved via more cumbersome RNA optimization.
In addition, the combination RTE/CTE has an added advantage in that it does not duplicate sequences, as is the case of vectors having multiple CTEs. This can be important, as the vectors are typically grown in bacteria and sequence duplication is often not well tolerated in many bacterial systems.
This invention thus provides vectors and methods of using the vectors to obtain high level expression of genes, including for applications such as gene therapy. Further, the RTE/CTE combination can be used in any gene transfer scenario where genes may be poorly expressed, e.g., DNA-based cytokine or viral antigen vaccine vectors. This applies to the field of retroviral vector development, the development of DNA-based vaccine vectors and other applications to enhance gene expression.